The ultimate goal of this research is to provide integrated systems that allow complex biological samples to be analyzed with high sensitivity and broad dynamic range. These systems are applicable to proteins, DNA and RNA oligomers, and metabolites from bodily fluids and tissues. This project is specifically focused on developing novel sample plates employing collimated hole structures that allow a variety of separation techniques to be efficiently coupled to MALDI-TOF and TOF-TOF mass spectrometry. Phase I established the basic feasibility of the new MALDI plates for interfacing with several widely used separation techniques including HPLC and slab gel electrophoresis, and with direct imaging of frozen tissue sections. Phase II will produce a new family of MALDI sample plates optimized for these applications and devices for interfacing with a variety of techniques for efficiently separating peptides, proteins, and metabolites. These devices integrated with new MALDI-TOF MS and MS-MS instruments being developed in parallel projects will provide orders of magnitude improvements in capabilities for global analysis of complex biological samples. Specific aims for Phase II are focused on practical MALDI sample plates employing collimated hole structures optimized for selected applications, and on practical devices for interfacing MALDI MS and MS- MS with established techniques for efficiently separating peptides, proteins, and metabolites. The definition of a practical CHS plate for these applications is that it must be sufficiently inexpensive to be disposable after single use, or there must be a simple, reliable procedure for cleaning the plates with no detectable sample carry-over. These new MALDI sample plates, parallel digestion plates, and the apparatus and reagents required for their use will provide important new products for commercialization in Phase III. In addition, complete integrated systems combining output of this project with results from other projects being pursued concurrently at Virgin Instruments Corp. will provide products with much larger potential markets. These include a fully automated LC-MALDI MS-MS system for global biological analyses;an automated "Molecular Scanner" for protein analyses using gels and MALDI MS and MS-MS;integrated global analysis laboratory;and an automated high-speed tissue imager. PUBLIC HEALTH RELEVANCE: The human genome project has established the molecular approach to understanding biology, but present knowledge of how an organism functions at the molecular level is really very poor. Functional analyses must be carried out, not only at the level of gene expression (transcriptomics), but also at the level of protein translation and modification (proteomics), and the metabolite network (metabolomics). Further improvements in chemistry, separations science, mass spectrometry, and bio-informatics will be required to complete this revolution. The interface between separations and mass spectrometry is believed to be a major bottleneck. The proposed research will provide important new tools toward overcoming this obstacle.